As described in the goals and objectives section of this report, this project consists of two specific aims: Isolation of novel genes regulating vascular integrity via forward genetics We use forward-genetic approaches to identify and characterize zebrafish mutants that specifically disrupt cranial vascular integrity in the zebrafish, with the aim of identifying novel candidate genes influencing the susceptibility to hemorrhagic stroke. We are carrying out an F2 genetic screen for ENU-induced dominant hemorrhage-prone mutants. We have screened over 500 genomes and identified 15 new vascular mutants showing increased susceptibility to intracranial hemorrhage. A bulked segregant mapping pipeline is in place to rapidly determine the rough position of newly identified mutants on the zebrafish genetic map, and fine mapping and molecular cloning is in progress for many of the mutants. We are also using exome sequencing and SNP analysis with a newly developed SNP database to perform higher-throughput cloning of mutants. These methods have led to the identification of the causative gene for approximately a third of the mutants thus far, and we are currently further characterizing the molecular nature of the defects in these mutants and continuing to identify the causative genetic defect in the other mutants. These new vascular integrity mutants promise to bring to light new pathways important in the maintenance of vascular barrier function. Functional analysis of zebrafish orthologs of human stroke disease genes Together with the Laboratory of Dr. Elizabeth Tournier-Lasserve, we are carrying out experimental analysis on zebrafish orthologs of human hemorrhagic stroke genes and/or genes identified as being in genetic pathways known to be involved in vascular integrity in humans. Our goal is to better understand the roles of these genes in maintaining vascular integrity by using the genetic and experimental tools available in the fish to probe their functions in vivo. Using new tools we have recently developed for analysis of vascular smooth muscle origins and function in the zebrafish, we are examining the effects on vascular integrity of defects in smooth muscle in addition to the endothelium.